BIOLOGY AND GENETICS OF ENTEROCOCCUS FAECALIS POLYSACCHARIDE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 肠球菌是医院获得性感染的主要原因，占所有医院感染的近10%。由于多重耐药性的存在，肠球菌引起的感染构成了一个重大的治疗挑战。拟议的研究将确定如何在遗传水平上实现胶囊多样性。最近的证据表明，粪肠球菌的衣壳血清型数量有限，大多数分离株属于4个主要血清型(A-D)。最近的临床证据表明，具有C型和D型包膜的粪肠球菌可能比表达其他包膜类型的菌株更具致病性。基于广泛的免疫交叉反应，血清型C和D似乎在结构上相关。C型包膜生物合成的遗传基础由9个基因的操纵子编码，命名为CPSC-K。C群和D群菌株之间的遗传比较显示，除cpsF外，操纵子中的每个基因都具有广泛的序列保守性，这只在C型菌株中存在。我们的目的是通过在粪肠球菌V583中构建cpsF的框内缺失突变体来解决这一差异。D型菌株与多拷贝质粒上天然囊膜启动子表达的cpsF的互补也将用于证实cpsF的存在赋予C型特异性的假设。这些衍生的菌株将用类型特异的抗血清进行酶联免疫吸附试验。此外，还将利用GC/MS对纯化的包膜材料进行碳水化合物组成分析，以确定C型和D型血清型差异的组成基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Entercocci are leading causes of hospital-acquired infections, accounting for nearly 10% of all nosocomial infections. Infections caused by enterococci constitute a significant treatment challenge due to the presence of multi-drug resistance. The proposed studies will determine how capsule diversity is achieved at the genetic level. Recent evidence suggests that E. faecalis has a limited number of capsular serotypes and that the majority of isolates fall into 4 predominant serotypes (A-D). Recent clinical evidence suggests that E. faecalis strains possessing capsule types C and D maybe more pathogenic than strains expressing other capsule types. Serotypes C and D appear to be structurally related based on extensive immunologic cross-reactivity. The genetic basis for serotype C capsule biosynthesis is encoded by an operon of 9 genes, designated cpsC-K. Genetic comparison between serogroup C and D strains revealed extensive sequence conservation for each gene in the operon with the exception of cpsF, which was only present in serotype C strains. We aim to address this difference by constructing an in-frame deletion mutant of cpsF in E. faecalis V583, a serotype C strain. Complementation of type D strains with cpsF expressed from the native capsule promoter on a multi-copy plasmid will also be used to confirm the hypothesis that the presence of cpsF confers type C specificity. These derived strains will be examined by ELISA using type-specific antisera. In addition, carbohydrate compositional analysis on purified capsular material using GC/MS will be performed to determine the compositional basis for the serotype differences between types C and D.